Fly strains
We used the following stocks: Oregon R (wild-type), nuf
1 [39], nuf
KG00314 (flybase), nuf
BRW [24], FRTG13::aPKC
K06403, baz
4
::FRT19a, FRT82B::crb
1, sec6
∆20
::FRTG13,/+ [40], sec5
E15
::FRT40A [26]. UAS-Rab11-RNAi (v22198) from the Vienna Drosophila RNAi Centre.
We used 69B-Gal4, 24B-Gal4, en-Gal4 and Hh-Gal4 as driver lines.
Immunohistochemistry
Imaginal discs were fixed for 20 minutes in paraformaldehyde 20 % followed by a second fixation of 20 minutes in paraformaldehyde 20 %–0.1 % TritonX-100.
The following primary antibodies were used: anti-Nuf (1:500, generated in this work as described in [11]), mouse and rabbit anti-aPKC (1:100 and 1:500, respectively) and rabbit anti-Myc (1:250) from Santa Cruz Biotechnology; mouse anti-Myc (Cell Signalling 1:500), rabbit anti-GFP (Molecular Probes, 1:300); anti-Dlg (1:100), anti-Crb (1:100) and anti-DECad (1:20) from Developmental Studies Hybridoma Bank, mouse anti-Myc (Cell Signalling, 1:500); anti-Rab11 (BD Biosciences, 1:100); anti-ßgal (Promega, 1:10.000), anti-Baz (1:500, this work), anti-DHC (1:500, [41]), anti-Rab5 (1:25, a gift from M. González-Gaitán) and anti-DPATJ (1:500, a gift from H. Bellen). Secondary antibodies were coupled to Alexa488, Alexa555 or Alexa647 (Molecular Probes).
Affinity-purified guinea pig antibody for Nuf was generated in this study by conventional methods. Briefly, E. coli BL21(DES)pLyS was transformed with pGEX-6P-1 Nuf FL and the protein overexpression was performed at 20 °C with 0.1 mM IPTG for 20 hours. The recovered cell pellets were lysed by sonication in PBS, 1 mM DTT and 1X complete protease inhibitors cocktail (Roche) buffer. After sonication, the lysate was incubated on ice with 1 % Triton-X100 for 30 minutes. The lysate was centrifuged (14,000 rpm, 4 °C, 30 minutes) and incubated with glutathione-Sepharose beads (Sigma) for 4 hours at 4 °C, the beads were washed three times with PBS and 1 mM DTT and twice with PBS, 1 mM DTT and 1 M NaCl. The protein bound to the beads was eluted with elution buffer (50mM Tris HCl pH 8.5, 0.1 %, Triton X-100, 100 mM NaCl, 1 mM DTT, 20 mM glutathione). The eluted material was lyophilised and sent to Genosphere Biotech to generate the antibody.
Affinity-purified rabbit antibody for Baz/Par-3 was generated in this study by transformation of BL21(DES)pLyS with pGEX-6P-1-Baz N1 [42] and following the same protocol as above with anti-Nuf.
Images were taken on a SP2-AOBS or a SPE Leica confocal microscopes and processed using FIJI and Adobe Photoshop programs.
Quantification of antibody staining and statistics
A total of fifteen confocal images of 0.16 μm thickness of a wing imaginal disc were projected using the average intensity algorithm (ImageJ) and five cells were randomly selected in the anterior and posterior compartments. The fluorescence intensity (pixel grey intensity shown in arbitrary units) was measured along a line 3 μm long. Different samples (n = 5) were measured for each genotype (n = 25).
In Additional file 4: Figure S4d the differences between fluorescence at the membrane and the cytoplasm were calculated for each cell analysed (n = 25) in anterior or posterior and in the different backgrounds as indicated. Data are displayed as a box plot and the statistical significance was calculated using the non-parametric Wilcoxon test.
To analyse Nuf distribution (Fig. 2i) 15 confocal images of 0.16 μm thickness of a wing imaginal disc expressing the different Nuf transgenes were projected using the average intensity algorithm (ImageJ). A total of five cells were randomly selected and five different samples were measured for each genotype (n = 25 each). The myc fluorescence intensity (pixel grey intensity shown in arbitrary units) was measured along a line 3 μm long that encompassed the two opposite membrane cells. Myc fluorescence was compared to PATJ, an apico-lateral protein of the Crb complex.
Figure 2k shows the fluorescence levels of myc-NufSA, measured as described above, in the border between two aPKC mutant cells (1) or two sibling wild type cells (2). The border cell was defined and measured by Par-3 levels. Keep in mind that Par-3 and GFP (marker of wild type cells) are stained in green indicating the higher basal levels of Par3 in wild type cells.
Pull-down assays and Western blots
To perform pull down experiments Nuf-GST fusion proteins were obtained from subclones in pGEX-6P-1 (Nuf FL) or pGEX2T (Nuf-NH and Nuf-CO). Purification was done as described in [11]. A total of 300 μg of protein extract from Drosophila embryos in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EGTA, 2 mM EDTA, 1 % Triton X-100, 5 mM DTT, 1 mM PMSF, 1 mM sodium orthovanadate, 1X complete protease inhibitor cocktail (Roche) and 10 mM β-glycerol phosphate) were incubated for 4 hours at 4 °C with 30 μg of glutathione-S-transferase (GST) or of the different GST-fusion proteins bound to glutathione beads as described in [10]. Complexes were washed three times using 50 mM Tris HCl pH 7.5, 50 mM NaCl and 0.1 % Triton X-100 and once with 50 mM Tris HCl pH 6.8. In vitro pulldown was performed incubating 20 μg of GST or Nuf-GST proteins bound to glutathione beads with 0.25 μg of PKCz (PRKCz Recombinant Human Protein, Life Technologies) in lysis buffer for 2 hours at 4 °C. Complexes were washed three times using PBS 1X supplemented with 0.5 M NaCl and once with 50 mM Tris HCl pH 6.8. The GST-tag of proteins expressed from pGEX-6P-1 plasmids was removed by PreScission protease (GE Healthcare) following the manufacturer’s protocol and used in vitro for the Rab11-GST pull-down assay as described in [10].
Expression and purification of the recombinant proteins GST-14-3-3ε and GST-DLIC was performed as described above for Nuf from pGEX-6P-1-14-3-3 and pGEX-6P-1-DLIC respectively.
Competitive pull-down assays
Kinesin and dynactin: 20 μg of GST, GST-NufNH wt, GST-NufNH S155A and GST-NufNH S155D fusion proteins were incubated with 300 μg of OR extract for 3 hours at 4 °C in lysis buffer (50 mM Tris HCl pH 7.5, 150 mM NaCl, 1 mM EGTA, 2 mM EDTA, 1 % Triton X-100, 5 mM DTT, 1 mM PMSF, 1 mM sodium orthovanadate, 1X complete protease inhibitor cocktail (Roche) and 10 mM β-glycerolphosphate). Beads were washed three times with 50 mM Tris HCl pH 7.5, 50 mM NaCl and 0.1 % Triton X-100 and incubated with 0, 50 or 200 μg of PKCz (PRKCz recombinant human protein, Life Technologies) for 1 hour at 4 °C. Beads were washed three times with IP15OO buffer and once with Tris HCl 50 mM pH 6.8.
GST-Rab11 and GST-DLIC: 20 μg of GST and GST-fusion proteins were incubated with 200 ng of NufFL wt, NufFL S155A and NufFL S155D proteins (GST was removed with Prescission protease, GE Healthcare) for 2 hours at 4 °C in Rab11 interaction buffer (PBS, 2 mM DTT, 1 mM sodium orthovanadate, 1 mM PMSF, 1X complete protease inhibitor cocktail) and DLIC interaction buffer (50 mM Tris HCl pH 7.5, 0.5 % Triton X-100, 150 mM NaCl, 10 % glycerol and 1 mM EDTA) respectively. Beads were washed three times with Rab11 interaction buffer supplemented with 50 mM NaCl for Rab11 assay and DLIC interaction buffer for DLIC assay. After that, beads were incubated with 0, 50 or 200 ng of PKCz in Rab11 interaction buffer or DLIC interaction buffer for 2 hours at 4 °C. Beads were washed three times with Rab11 interaction buffer supplemented with 50 mM NaCl or DLIC interaction buffer. In both cases, beads were finally washed with Tris HCl 50 mM pH 6.8.
Western blotting was done according to standard procedures using guinea pig anti-Nuf (1:5000), rabbit anti-aPKC (Santa Cruz Biotechnologies, 1:5000), anti-GST (Santa Cruz Biotechnologies, 1:3000), mouse anti-Flag (Sigma, 1:10000), rabbit anti-Kinesin (Cytoskeleton, 1:3000), anti-Glued (Dynactin, 1:1000 [43]), rabbit anti 14-3-3 (Santa Cruz Biotechnologies, 1:500) and mouse anti-α-Tubulin (Sigma, 1:10000).
aPKC phosphorylation assay
This method has been adapted from [44]. For each reaction, 150 ng of recombinant substrate (GST-fusion proteins) was incubated with 140 ng of human recombinant PKCiota or PKCz produced in Sf9 cells (Moscat laboratory) in phosphorylation buffer {(35 mM Tris HCl pH 7.5, 10 mM MgCl2, 0.1 mM CaCl2, 0.5 mM EGTA, 1 mM DTT and 0.1 mM ATPγS (Biolog)}) for 1 hour at 30 °C. The reaction was stopped by adding 20 mM EDTA and samples were incubated with 2.5 mM PNMB (Abcam) for 1 hour at room temperature. The samples were analysed by SDS-PAGE and western blotting using anti-thiophosphate ester antibody (Abcam, 1:5000). For MS/MS detection of Nuf phosphopeptides, an in-vitro kinase assay was performed using 10 μg of GST-Nuf FL and 10 μg of PKCiota in 400uM ATP and 1x phosphorylation buffer. Phosphorylation products were digested and processed using LS-MS/MS analysis by the SBMRI Proteomic Service.
Phosphorylated Nuf −14-3-3ε pull-down assay
A total of 20 μg of GST and GST-NufFL wt were incubated for 1 hour at room temperature with phosphorylation buffer adding ATP and 500 μg of PKCz only to the Nuf phosphorylated experiment. Beads were washed three times with 14-3-3 interaction buffer (25 mM Hepes pH 7.5, 12.5 mM MgCl2, 20 % glycerol, 0.1 % NP40, 150 mM KCl, 1 mM DTT) and were incubated with 500 ng of eluted 14-3-3ε (removing GST with Prescission protease) in 14-3-3 interaction buffer adding 30 μg of BSA for 3 hours at 4 °C. After that, beads were washed three times with 20 mM Tris HCl pH 8, 100 mM NaCl, 1 mM EDTA and 0.5 % NP40, and once with Tris HCl 50 mM pH 6.8.
Additional Methods, including primers used, are given in Additional file 7.